Different treatment response to systemic corticosteroids according to white blood cell counts in severe COVID-19 patients

Abstract Background Limited data are available in COVID-19 patients on the prediction of treatment response to systemic corticosteroid therapy based on systemic inflammatory markers. There is a concern whether the response to systemic corticosteroid is different according to white blood cell (WBC) counts in COVID-19 patients. We aimed to assess whether WBC count is related with the clinical outcomes after treatment with systemic corticosteroids in severe COVID-19. Methods We conducted a retrospective cohort study and analysed the patients hospitalised for severe COVID-19 and received systemic corticosteroids between July 2020 and June 2021. The primary endpoint was to compare the composite poor outcome of mechanical ventilation, extracorporeal membrane oxygenation, and mortality among the patients with different WBC counts. Results Of the 585 COVID-19 patients who required oxygen supplementation and systemic corticosteroids, 145 (24.8%) belonged to the leukopoenia group, 375 (64.1%) belonged to the normal WBC group, and 65 (11.1%) belonged to the leukocytosis group. In Kaplan-Meier curve, the composite poor outcome was significantly reduced in leukopoenia group compared to leukocytosis group (log-rank p-value < 0.001). In the multivariable Cox regression analysis, leukopoenia group was significantly associated with a lower risk of the composite poor outcome compared to normal WBC group (adjusted hazard ratio [aHR] = 0.32, 95% CI 0.14–0.76, p-value = 0.009) and leukocytosis group (aHR = 0.30, 95% CI = 0.12–0.78, p-value = 0.013). There was no significant difference in aHR for composite poor outcome between leukocytosis and normal WBC group. Conclusion Leukopoenia may be related with a better response to systemic corticosteroid therapy in COVID-19 patients requiring oxygen supplementation. KEY MESSAGES In severe COVID-19 treated with systemic corticosteroids, patients with leukopoenia showed a lower hazard for composite poor outcome compared to patients with normal white blood cell counts or leukocytosis. Leukopoenia may be a potential biomarker for better response to systemic corticosteroid therapy in COVID-19 pneumonia.


Introduction
Systemic corticosteroid therapy has been a critical component for the management of severe coronavirus disease 2019 (COVID-19) with respiratory failure [1]. In previous randomised controlled trials (RCTs), administration of systemic corticosteroids improved mortality and increased ventilator free days in COVID-19 patients [2,3]. Especially, dexamethasone had significant benefits in the COVID-19 patients who required oxygen supplementation or mechanical ventilation [2]. However, the mortality rate of severe COVID-19 patients was still as high as 26.6%, even though systemic corticosteroids were used [1]. Therefore, we need to differentiate the COVID-19 patients with heterogeneous responses to systemic corticosteroids.
To provide precise and individualised management for the COVID-19 patients, it is essential to identify the subgroups who either responds well or poorly to standard treatment consisting mainly of systemic corticosteroids. Considering that the efficacy of corticosteroids on COVID-19 relies on their anti-inflammatory or immunomodulatory effects [4], systemic inflammatory biomarkers may have a potential role to predict the response to corticosteroids. There have been few studies that investigated inflammatory phenotypes related with the response to systemic corticosteroids. A recent study showed that systemic corticosteroids may be less effective in the critically-ill COVID-19 patients with hypo-inflammatory phenotypes [5]. A better clinical response to systemic corticosteroids has been expected in hyper-inflammatory phenotypes than hypo-inflammatory phenotypes [6]. Among various systemic inflammatory markers, white blood cell (WBC) has been considered as an important indicator to differentiate hyper-and hypo-inflammatory phenotypes in acute respiratory distress syndrome (ARDS) [7,8]. WBC count was related with cytokine storm syndrome, higher severity, and poor prognosis in COVID-19 [9][10][11]. Although WBC count is an important biologic marker for systemic inflammation, it has not been figured out whether treatment response to systemic corticosteroid for severe COVID-19 is different according to WBC count. Therefore, we investigated whether the clinical outcomes after treatment with systemic corticosteroid are different according to the WBC counts in COVID-19 patients who required oxygen therapy.

Methods
We reported the present study following the guidance of the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) statement.

Study design and participants
We conducted a retrospective cohort study and analysed the patients who were hospitalised for severe COVID-19 at Boramae Medical Centre between July 2020 and June 2021. All the eligible patients were: (1) age >18 years old, (2) diagnosed with COVID-19 through quantitative reverse transcription-polymerase chain reaction (RT-qPCR) assays using respiratory tract samples, (3) treated with systemic corticosteroids for severe COVID-19. Systemic corticosteroids were administrated when the COVID-19 patients required oxygen therapy or had a significant desaturation (room-air percutaneous arterial oxygen saturation [SpO 2 ] < 90%). Exclusion criteria were immunosuppressive conditions including haematologic malignancy or current chemotherapy for active cancer, administration of systemic corticosteroids or immunosuppressive drugs for other conditions, pregnant status, and terminal stage disease with documented Do Not Resuscitate order.

Study outcomes
The primary endpoint was to compare the composite poor outcome including mechanical ventilation, ECMO and mortality among three groups classified based on different WBC counts: leukopoenia, normal leukocyte, and leukocytosis groups. The patients with WBC <4000 were defined as leukopoenia group, those with WBC from 4000 to 9999 were defined as normal WBC group, and those with WBC !10,000 were defined as leukocytosis group.

Statistical analyses
Demographic information, symptomatic manifestation, vital signs and parameters for oxygen status, laboratory and radiologic results, treatments and clinical outcomes were compared among the different three groups. We analysed categorical variables using Pearson's chi-squared test or the Fisher's exact test and continuous variables using ANOVA test or Kruskal-Wallis test. To analyse linear trend among the three groups, the Cochran-Armitage test was used for categorical variables, and linear regression analysis was used for continuous variables. We conducted repeated-measure ANOVA tests to evaluate the changes of WBC counts. We performed survival analysis with Kaplan-Meier estimation. To minimise the potential risk of bias due to confounding factors, multivariate Cox regression analysis was conducted with statistically significant covariables selected through backward elimination. Statistical significance was determined as p-values <0.05. All the statistical analyses were conducted using R statistical software, version 4.1.1 (R Core Team, [2021], Vienna, Austria).

Ethics
This study was conducted in accordance with the principles of the Declaration of Helsinki. The Institutional Review Board of Seoul Metropolitan Government-Seoul National University Boramae Medical Centre approved the study protocol waived the need for informed consent for access to the electronic medical records (IRB No. 30-2022-40).
The baseline characteristics of the included patients are presented in Table 1. Leukocytosis group was older with mean age of 74.2 years compared to 68.1 and 68.5 years in leukopoenia and normal WBC group, respectively. The proportion of hypertension and asthma showed a positive linear trend according to the WBC status. Otherwise, there were no significant differences across the groups.

Clinical characteristics and initial laboratory indices
The clinical characteristics and initial laboratory indices are presented in Table 2. For initial vital signs and parameters, SBP, HR, and oxygen requirement or FiO 2 showed positive linear trends according to the WBC status while SpO 2 , SF ratio, and ROX index showed negative linear trends. For initial laboratory indices, neutrophil-to-lymphocyte ratio (NLR) showed a positive linear trend according to the WBC status while monocytes showed a negative linear trend. Platelet counts, CRP, procalcitonin, LDH, D-dimer, BUN, AST, ALT, total bilirubin, and PT INR also showed positive linear trends. In the analysis of WBC changes during 5 days of steroids, WBC count and neutrophil count increased in leukopoenia and normal WBC group, while decreased in leukocytosis group (supplemental Figure  S1). In addition, lymphocyte count of leukopoenia group was more increased compared with that of leukocytosis group, although lymphocyte counts at D1 were similar. There was no difference in the presence of pneumonic infiltration in chest X-rays across the groups.

Treatment and clinical outcomes for COVID-19
Treatment of COVID-19 infection across the groups showed difference in the duration of steroid therapy; it was the longest in leukocytosis group with the mean of 12.5 days compared to 9.5 days in both leukopoenia and normal WBC group ( Table 3). The use of remdesivir or antibiotics was similar across the groups. The hospital length of stay was the longest in leukocytosis group with 31.3 days compared to 16.7 days and 15.7 days in leukopoenia and normal WBC group, respectively. In Kaplan-Meier curve, the composite poor outcome of mechanical ventilation, ECMO and mortality was significantly reduced in leukopoenia group compared to leukocytosis group (log-rank pvalue < 0.001), but not compared to normal WBC group (log-rank p-value ¼ 0.060) (Figure 2).

Multivariable analysis to evaluate risk factors of composite poor outcome
In the univariable Cox regression analysis, leukopoenia group showed a lower hazard ratio (HR) for composite poor outcome compared to normal WBC group. In addition, age greater than 65 years, connective tissue disease, febrile manifestation, a higher systolic blood pressure, procalcitonin greater than 0.5 ng/mL, a lower ROX index, neutrophilia (!7500, 103/mL), NLR, a lower haemoglobin, and a lower platelet count were associated with a higher HR for composite poor outcome. In the multivariable Cox regression analysis, leukopoenia group was significantly associated with a lower risk of composite poor outcome compared to normal WBC group (adjusted HR (aHR)¼0.32, 95% CI ¼ 0.14-0.76, p-value ¼ 0.009) and leukocytosis group (aHR ¼ 0.30, 95% CI ¼ 0.12-0.78, p-value ¼ 0.013) in COVID-19 patients who were treated with systemic corticosteroids (Table 4). On the other hand, leukocytosis group was not associated with composite poor outcome (aHR 1.08, 95% CI 0.52-2.21, p-value ¼ 0.845) compared to normal WBC group. In the multivariable analyses according to the differential count of white blood cells, neutrophilia was related with a higher risk of composite poor outcome, but there was no association between neutropenia or lymphopenia and composite poor outcome (supplemental Table S3 and S4). In addition, we found no association between NLR and composite poor outcome in the multivariable analysis (supplemental Table S5). Nomogram of variables on composite poor outcome is described in supplemental Figure S2.

Discussion
Our study investigated treatment response of systemic corticosteroid therapy in COVID-19 patients who   <0.001 0.012 a Dose is based on an equivalent dose of methylprednisolone. Data are presented as n (%) or mean ± standard deviation. Ã Categorical variables were analysed using Cochran-Armitage test, continuous variables were analysed using linear regression.
ECMO: extracorporeal membrane oxygenation. required oxygen therapy according to the blood leukocyte counts. Leukopoenia group showed a significantly lower hazard of composite poor outcome consisting of mechanical ventilation, ECMO and mortality compared to normal WBC or leukocytosis group in multivariable analysis. Leukocytosis group showed an increased risk of composite poor outcome in survival analysis, but did not increase the hazard of composite poor outcome compared to normal WBC group after adjustment for disease severity. Neutrophilia was related with an increased hazard of composite poor outcome, while neutropenia, lymphopenia, and NLR were not associated with composite poor outcome in multivariable analyses. Therefore, a better treatment response may be expected in the patients with leukopoenia who were treated with systemic corticosteroids for severe COVID-19 pneumonia.
There has been a growing interest in identifying clinical sub-phenotypes of COVID-19 pneumonia or COVID-19-related ARDS for establishing individualised therapy. Especially, phenotyping with various inflammatory profiles has been expected useful in predicting treatment response to systemic corticosteroid in COVID-19. A previous study conducted cluster analysis using inflammatory biomarkers to investigate hyper-and hypoinflammatory phenotypes in critically ill COVID-19 patients who used systemic corticosteroids [5]. This study reported that a better efficacy of systemic corticosteroid therapy was observed in the patient group with hyper-inflammatory phenotypes. Although systemic corticosteroid treatment was not randomised, another study conducted latent class analysis to find a better response to systemic corticosteroid in the patients with hyper-inflammatory phenotypes [8]. COVID-19 can cause systemic hyper-inflammation with features of cytokine storm syndrome [12,13], and systemic corticosteroids are thought to be effective because they down-regulate systemic hyper-inflammatory response.
Our study figured out that there may be a difference in the response to systemic corticosteroids according to the WBC profiles, which is one of the important inflammatory markers. WBC count reflected the immunologic response in COVID-19 that was different from other respiratory viral infections and was related to the prognosis in COVID-19 patients. The leukocyte expression profiles were different between hypo-and hyper-inflammatory ARDS [14]. In our study, the hazard of composite poor outcome was lower in leukopoenia group compared to normal WBC or leukocytosis group in severe COVID-19 patients who used systemic corticosteroid. Leukopoenia has been frequently found in the hyperinflammatory syndromes including secondary haemophagocytic lymphohistiocytosis, macrophage activation syndrome, and cytokine release syndrome [15]. Therefore, leukopoenia may be a biologic marker for hyper-inflammatory phenotype in COVID-19 with a better response to systemic corticosteroid [16][17][18].
Lymphopenia has been suggested as a poor prognostic factor in COVID-19. Compared to the cytokine storm syndromes caused by other diseases, COVID-19 induced hyper-inflammation is different in its haematologic manifestation as decreased lymphocyte count was related with poor outcomes [19,20]. The mechanism behind the development of lymphopenia in COVID-19 is suggested to be multi-factorial: direct cell infection by the virus, indirect destruction by hyperinflammation caused via IL-6 and TNF-a pathways, and lymphocytes recruitment to the inflammatory lung tissues [21]. Our study showed that lymphopenia was not a prognostic factor in COVID-19 patients who used systemic corticosteroids. Systemic corticosteroids are reported to increase lymphocyte counts in clinically responsive COVID-19 patients [22]. Immunomodulating effect of systemic corticosteroids may recover T-cell dysregulation and or alleviate the inflammatory process [23,24].
Neutropenia was only found in leukopoenia group but neutrophilia was not. Low neutrophil count was related with a higher mortality, and there was an insignificant trend of decreasing mortality after administration of systemic corticosteroid in COVID-19 patients with neutropenia [25]. In our study, neutropenia was not significantly associated with the hazard of composite poor outcome after treatment with systemic corticosteroid. Neutrophilia is another haematologic marker associated with poor outcome in COVID-19 [26][27][28]. Neutrophilia is related with the increased release of proinflammatory cytokines, activation of neutrophil, induction of oxidative stress, and formation of neutrophil extracellular traps, resulting in inflammatory lung injury [29]. Neutrophilia has been a predictive marker of progression of ARDS associated with a systemic hyper-inflammatory response [29]. However, even though systemic corticosteroid was administered, the patients with neutrophilia showed a higher hazard of the composite poor outcome in our study. Therefore, additional therapeutic intervention combined with systemic corticosteroid may be necessary to further modify dysregulation of excessive inflammatory response to COVID-19 pneumonia in the patients with neutrophilia. The present study has several limitations. First, our study is a retrospective cohort study in a single medical centre. For generalisation, external validation within a larger cohort is needed. Second, different prognosis according to WBC counts may be related with a different severity of COVID-19, rather than the response to systemic corticosteroid. Therefore, we included only severe COVID-19 patients who required oxygen therapy and analysed them with adjustment for age, comorbidities, ROX index, and other laboratory findings. Even considering this limitation, lymphopenia is speculated as a biologic indicator for favourable outcomes in COVID-19 patients treated with systemic corticosteroid. Third, to properly estimate the different efficacy of systemic corticosteroid according to WBC counts, clinical outcomes should be compared according to whether or not steroids were administered in each group. However, it is not feasible in real-world to compare prognosis between the patients who used steroids and those who did not because of the considerable difference in disease severity between them. In addition, random allocation for steroid administration according to specific biomarkers in the patients with severe COVID-19 who requires oxygen is considered ethically violated under current treatment consensus. Therefore, our study evaluated clinical outcomes as an indirect indicator for treatment response to systemic corticosteroids in severe COVID-19 patients. Fourth, further in vivo or in vitro study is needed to elucidate the pathophysiological background behind WBC changes with corticosteroid therapy during severe COVID-19 infection.

Conclusion
Leukopoenia group showed a lower hazard for composite poor outcome compared to normal WBC or leukocytosis group in severe COVID-19 patients who were treated with systemic corticosteroids. Leukopoenia may be a potential biomarker for better response to systemic corticosteroid therapy in COVID-19 pneumonia.